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Correspondence to Niek de Vries, Department of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam 1100 DE, The Netherlands; n.devries{at}amc.uva.nl

Abstract

Background The onset of seropositive rheumatoid arthritis (RA) is preceded by the presence of specific autoantibodies in the absence of synovial inflammation. Only a subset of these at-risk individuals will develop clinical disease. This impedes efforts to implement early interventions that may prevent onset of clinically manifest disease. Here we analyse whether clonal changes in the B cell receptor (BCR) repertoire can reliably predict onset of signs and symptoms.

Methods In a prospective cohort study in 21 individuals at risk for RA based on the presence of autoantibodies, the BCR repertoire of paired peripheral blood and synovial tissue samples was analysed using next-generation BCR sequencing. BCR clones that were expanded beyond 0.5% of the total repertoire were labelled dominant. The relative risk (RR) for onset of arthritis was assessed using the presence of ≥5 dominant BCR clones as cut-off. Findings in peripheral blood were validated in an independent prospective cohort of 50 at-risk individuals. Based on the test cohort, individuals in the validation cohort were considered positive if peripheral blood at study entry showed ≥5 dominant BCR clones.

Findings Both in the test and validation cohort, the presence of ≥5 dominant BCR clones in peripheral blood was significantly associated with arthritis development after follow-up (validation cohort RR 6.3, 95% CI 2.7 to 15, p<1×10−4). Even when adjusted for a recently described clinical prediction rule the association remained intact (RR 5.0, 95% CI 1.2 to 20, p=0.024). When individuals developed arthritis, dominant BCR clones disappeared from peripheral blood and appeared in synovial tissue, suggesting a direct role of these clones in disease pathogenesis.

Interpretation Dominant BCR clones in peripheral blood predict onset of clinical signs and symptoms of RA in at-risk individuals with high accuracy. Our data suggest that during onset of RA these clones shift from peripheral blood to the target tissue.

arthritis

B cells

early rheumatoid arthritis

synovitis

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Introduction

Rheumatoid arthritis (RA) is a prototypic chronic autoimmune disease with partly unknown aetiology. Clinically manifest arthritis due to synovial inflammation is the hallmark feature of RA. However, it is not the first sign of disease, as patients may already experience arthralgia and the development of synovial inflammation may be preceded by the presence of disease-specific autoantibodies.1–3 This situation is reminiscent of that in several other immune-mediated inflammatory diseases.4–7

RA-specific autoantibodies, IgM-rheumatoid factor (RF) and/or anticitrullinated protein antibodies (ACPA), can be present up to 15 years before onset of disease.1 8 9 Towards the onset of clinically evident arthritis the ACPA repertoire may broaden due to epitope spreading,10 11 and levels of inflammatory cytokines and chemokines may increase.12 13 Although the presence of ACPA is highly specific for RA14 and may precede its onset, only 20% of the autoantibody positive subjects will develop arthritis within 4 years.15

The presence of these autoantibodies preceding the development of RA clearly points to a role for B cells and plasma cells in the pathogenesis of RA. The pathogenic role of B cells in established RA is supported by the known association with autoantibodies,16 marked infiltration of the synovium by B cells and plasma cells,17 the production of autoantibodies in the synovial compartment18 and the response to B cell-depleting therapy.19 Consistent with this notion, B-cell receptor (BCR) repertoire analysis showed that dominant clones were found in the inflamed synovial tissue of patients with established RA.20

We hypothesised that dominant clones may be detected by BCR sequencing in the peripheral blood during the preclinical phase of RA. This might help predict which at-risk individuals will develop arthritis over time. We tested this hypothesis analysing paired peripheral blood and synovial tissue samples from individuals at risk for developing RA in a prospective cohort study. We found that the presence of dominant peripheral blood BCR clones can predict future onset of RA, and we validated these findings in an independent cohort. Of interest, during the transition to clinically manifest arthritis the BCR clones were not traceable in peripheral blood anymore, but they were found in synovial tissue as highly dominant clones, pointing to a shift of BCR clones to the synovial compartment. The observation that dominant peripheral blood BCR clones can predict future onset of disease may be relevant for other B cell-mediated autoimmune diseases as well.

Methods

Study subjects

Sixty-five consecutive individuals without arthritis, but at risk for the development of RA defined by the presence of IgM-RF and/or ACPA (anti-CCP2 test, Eurodiagnostica), were prospectively followed (further denoted as ‘at-risk individuals’).2 21 From the 65 included individuals, we randomly selected 10 autoantibody positive at-risk individuals who did not develop arthritis (median follow-up 69 (range 42–78) months), and 11 individuals who did develop arthritis (median follow-up 15 (range 0–65) months) as test cohort. Nine individuals of the latter group fulfilled the 2010 ACR/EULAR criteria for RA at onset of arthritis,22 23 while two had unclassified arthritis at the moment of development of arthritis but subsequently did fulfil RA criteria over time. In addition, 10 autoantibody negative healthy individuals without any joint complaints were included as controls (clinical characteristics of all three groups described in table 1 and online supplementary table S1).

Supplementary Material

Clinical characteristics of healthy controls, at-risk individuals who did not develop arthritis over time and at-risk individuals who developed arthritis. At-risk individuals have elevated titres for IgM-RF (>12.5 kU/L) and/or anti-CCP (>25 kAU/L). Healthy individuals have low titres for IgM-RF (≤12.5 kU/L) and anti-CCP (≤25 kAU/L)

In total, 21 at-risk individuals and 10 healthy controls were included in this part of the study.

A validation cohort was used consisting of 50 consecutively included individuals with elevated ACPA and/or IgM-RF without any signs of arthritis and at least 36 months follow-up (further details are described in ref 24). During sequencing and bioinformatic analysis for dominant clones laboratory personnel was blinded for clinical data and outcome.

The cohort studies were approved by the local medical ethical committees of the Academic Medical Center/University of Amsterdam and MC Slotervaart Amsterdam, and all study subjects gave written informed consent.

Peripheral blood and synovial tissue sampling and processing

In the 21 at-risk individuals of the test cohort, mini-arthroscopic synovial biopsy sampling was performed upon inclusion in a (non-arthritic) knee joint as previously described.25 Peripheral blood samples were drawn and stored in PAXGene Blood RNA tubes according to the manufacturer’s instructions (catalogue #762165, PreAnalytiX, Breda, the Netherlands). Storage of synovial biopsies, isolation and quantification of RNA, and cDNA synthesis were performed as described previously.26 Mini-arthroscopy in at-risk individuals who subsequently developed arthritis was performed on the same joint, after patients fulfilled the 2010 ACR/EULAR criteria for RA22 23 and before initiation of treatment.

Linear amplification and next-generation sequencing (NGS)

The linear amplification protocol has been extensively described before.26 Details are provided in the online supplementary methods. Samples were prepared for next-generation sequencing according to the manual for amplicon sequencing, and sequenced on a Roche Genome Sequencer FLX (Titanium platform). 10,000 BCRheavy sequences were analysed for each peripheral blood sample and 7500 BCRheavy sequences for each synovial tissue sample. We use the term dominant BCR clone to denote clones whose unique BCR signals represent ≥0.5% of the repertoire, as described earlier.20

Bioinformatics pipeline and data analysis

The bioinformatics pipeline used to obtain the BCR sequences was described previously in detail27 and contains four modules: multiplex identifier sorting, identification of V and J gene segments, CDR3 detection and removal of artefacts. Immunoglobulin isotype homology was determined using the National Center for Biotechnology Information’s open-access web tool Megablast and reference sequences for the human immunoglobulin heavy-chain constant regions, allowing a sequence homology >97%.28 Values are expressed as mean and SD or median and IQR range, according to criteria for (non-)parametric analysis. Differences between groups were analysed using Student’s t-test, Mann-Whitney U test, one-way analysis of variance or χ2 test where appropriate. Receiver operating characteristic (ROC) curves were used to determine cut-off values for the prediction of arthritis development in the test cohort. Logistic regression analysis was used to predict the added value of high-throughput fingerprinting and quantitation of BCR clones compared with an existing prediction model for the development of RA.24 GraphPad Prism software version 6 and PASW Statistics version 22 were used to perform the analyses. p-values <0.05 were considered statistically significant.

Results

Based on earlier observations that dominant BCR clones are present in the synovial tissue during clinically manifest RA, we hypothesised that these clones might be detectable in the peripheral blood before development of arthritis. Indeed, multiple dominant BCR clones were detected in peripheral blood of all 11 prospectively followed at-risk individuals who developed arthritis from the test cohort, as long as 66 months before the clinical onset of arthritis. In contrast, dominant BCR clones were nearly absent both in the 10 at-risk individuals who did not develop arthritis and in the 10 healthy individuals (figure 1A). We observed that the number of dominant BCR clones, the impact of all dominant BCR clones combined (size of all dominant clones combined as percentage of the total repertoire) and the impact of the most dominant BCR clone were increased in at-risk individuals who developed arthritis during follow-up, compared with those who did not develop arthritis and healthy individuals (number of dominant clones mean 9.7±8.0 vs 0.8±0.8 vs 0.7±0.7, respectively, p=0.001 (figure 1B), impact of the dominant clones median 16.4% of the total repertoire, IQR 3.7%–33.7% vs 0.7% IQR 0%–1.7% vs 0.5% IQR 0%–1.1%, respectively, p<0.0001 (figure 1C) and impact of the single most dominant clone mean 5.5%±4.6% vs 0.7%±0.7% vs 0.6%±0.4%, respectively, p<0.0003 (figure 1D)). Subsequently, we analysed synovial tissue biopsies in at-risk individuals obtained during the preclinical phase, but these samples contained BCR mRNA levels that were too low to allow next-generation sequenting (NGS). The low BCR mRNA levels in the synovium during the preclinical stage of the disease are explained by the absence of B cell infiltration as demonstrated before using immunohistochemistry.3

(A) Scatterplot of the BCR repertoire in peripheral blood of 11 at-risk individuals who developed arthritis (at-risk - developed arthritis), 10 at-risk individuals who did not develop arthritis (at-risk - no arthritis developed) and 10 autoantibody negative healthy individuals. Each dot represents one clone. The size of the clones is depicted as percentage of the total BCRheavy sequences. (B) The absolute number of dominant BCR clones (clonal size ≥0.5% of the total repertoire), (C) the impact of all dominant clones combined and (D) the size of the single most dominant clone, in at-risk individuals who developed arthritis (at-risk arthritis, n=11) versus at-risk individuals who did not develop arthritis yet (at-risk no arthritis, n=10), and healthy individuals (healthy, n=10). Bars show mean and SD, ***p<0.0001, **p<0.001 using one-way ANOVA. ANOVA, analysis of variance; BCR, B-cell receptor.

Collectively, these observations demonstrate that dominant BCR clones are readily detectable in peripheral blood during the preclinical phase in all at-risk individuals who will develop RA after follow-up, but not in those who did not.

The presence of dominant BCR clones predicts future arthritis development

Having shown that the presence of BCR clones can be detected in peripheral blood in all at-risk individuals who will subsequently develop RA, in some cases after several years, we next aimed to develop a biomarker that can be used to identify individuals who have a high risk of developing arthritis in the short term. Such patients might be treated in the at-risk phase to prevent onset of arthritis.29 A clinically relevant follow-up period of 36 months was chosen to evaluate arthritis development. This time period may carry a risk high enough to justify preventive pharmacological intervention, while being short enough to infer urgency for treatment.

We designed three tests based on the number of dominant BCR clones present, the impact of all dominant clones combined on the BCR repertoire and the impact of the single most dominant BCR clone; ROC curves are depicted in figure 2A–C. Based on these ROC curves, optimal cut-offs were determined at ≥5 dominant BCR clones in peripheral blood, a combined impact ≥3.7% and an impact of the most dominant clone ≥2.5%, respectively. We decided to use the presence of ≥5 dominant BCR clones as comprehensible and intuitive marker for further studies. This is further denoted as ‘BCR-clone positive,’ and <5 dominant BCR clones as ‘BCR-clone negative,’ and collectively as the BCR-clone model.

Receiver operating characteristic curves for (A) the number of dominant clones, (B) the impact of all dominant clones combined and (C) the impact of the most dominant clone in at-risk individuals (n=21). The development of arthritis was analysed after 36 months of follow-up. The arrow points to the cut-off value chosen, and the corresponding value is shown. (D) Kaplan-Meier curve for BCR-clone positive and BCR-clone negative individuals in the test cohort, assuming the at-risk individuals analysed represent a random selection of the total at-risk individuals (n=65). (E) Kaplan-Meier curve for BCR-clone positive and BCR-clone negative individuals in the validation cohort. (F) Table describing sensitivity, specificity, PPV and NPV including 95% CIs for the BCR-clone model, in the test cohort and the validation cohort. AUC, area under the curve; BCR, B-cell receptor; NPV, negative predictive value; PPV, positive predictive value.

The cut-off of ≥5 dominant clones in peripheral blood resulted in two clearly distinguishable groups, and corresponding sensitivity of 78%, specificity of 92%, positive predictive value (PPV) of 72% and negative predictive value (NPV) of 94% (figure 2D, 2F for the Kaplan-Meier curve, log rank test p<0.001). We had access to a second independent cohort of 50 subjects to validate our findings using the same cut-off value. Fifteen at-risk individuals developed arthritis within 36 months; the characteristics are described in table 2.

The 50 at-risk individuals in the validation cohort were previously used to develop a prediction model for the development of RA,24 the risk rule model. This describes a composite score of multiple clinical parameters categorising at-risk individuals into low, intermediate and high-risk individuals (respectively 17, 20 and 13 individuals). Using logistic regression analysis to calculate the added value of the BCR-clone model to the existing risk rule, an overall relative risk of 5.0 (95% CI 1.2 to 20, p=0.024) was found. In the low, intermediate and high-risk groups the relative risk contributed by BCR-clone positivity was estimated at 18 (95% CI 0.6 to 520), 6.1 (95% CI 1.9 to 20) and 1.2 (95% CI 0.6 to 2.7), respectively.

In conclusion, we show that at-risk individuals with five or more dominant BCR clones in peripheral blood have an 83% risk of developing arthritis within 36 months, compared with a risk of 13% in individuals with four or less dominant BCR clones. Moreover, analysis after 5 years revealed that all individuals who initially tested positive developed arthritis.

Dominant BCR clones present in peripheral blood during the preclinical phase have migrated to synovial tissue in clinically manifest RA

We hypothesised that if the observed dominant BCR clones are involved in synovial inflammation, then these clones might also be detectable in synovial tissue after onset of RA. To this end, we analysed peripheral blood samples obtained during the preclinical stage, and paired blood and synovial tissue after onset of arthritis in eight individuals who developed arthritis.

Up to 29.3% (median, IQR 9.9%–45.8%) of all preclinically detectable dominant peripheral blood BCR clones were detectable in synovial tissue after development of arthritis. All ranked within the top 25 most dominant clones in synovial tissue (figure 3A–C). Most strikingly, none of the preclinically detectable dominant peripheral blood BCR clones could be recovered from peripheral blood after arthritis developed (figure 3B,C). Additional analyses of the dominant clones found in both peripheral blood and synovial tissue showed that these clones are class switched to the IgG1 isotype and enriched for IGHV4-34, features that are all associated with autoreactivity, while IGHV3-23, IGHV1-69, CDR3 length, ACPA or IgM-RF titres, and HLA-DRB1 shared epitope positivity (SE) are comparable between the three groups (described in online supplementary results, supplementary figures 2 and 3, and supplementary table 2).30–33

Scatterplot of the BCR repertoire in synovial tissue (A) and peripheral blood (B) after arthritis development in eight patients. Each dot represents one clone. Clones in red represent clones that were dominantly present in peripheral blood during the preclinical phase. The size of the clones is depicted as percentage of the total BCRheavy sequences. (C) Dot plot showing the overlap between dominant BCR clones in the preclinical phase and after arthritis development (n=8). The y-axis depicts the rank of the clones found in blood during the preclinical phase (all eight patients pooled). On the left x-axis the overlap with dominant clones in peripheral blood after arthritis development, on the right x-axis the overlap with dominant clones in synovial tissue after arthritis development. In case no overlap was found, the dots were marked ‘no overlap.’ BCR, B-cell receptor; PB, peripheral blood; ST, synovial tissue.

Together, these analyses show that dominant BCR peripheral blood clones present during the preclinical phase of arthritis are in part retrievable as dominant clones in synovial tissue once arthritis becomes clinically apparent. At this time point, the clones are no longer found in peripheral blood anymore. These migratory clones have features that have been associated with autoreactivity.

Discussion

The results presented here show that the presence of dominant BCR clones in peripheral blood predicts with high accuracy the onset of arthritis in patients who are at risk of developing RA. Moreover, we found support that these dominant clones may migrate to synovial tissue once arthritis becomes apparent. These findings are consistent with the notion that B-cell abnormalities occur up to several years before the onset of synovial inflammation, and that the development of RA is a multistep process. Conceivably, a ‘second hit,’ for instance a trauma or viral infection, may lead to synovial inflammation, subsequent migration of autoreactive B-cell clones towards the synovium and impaired resolution of inflammation in patients with pre-existing systemic autoimmunity.2 34 This work provides the rationale for future studies on B-cell abnormalities during the preclinical stage in other immune-mediated inflammatory diseases like systemic lupus erythematosus (SLE), multiple sclerosis (MS) and type 1 diabetes (T1D), and opens up the perspective of preventive intervention.

Since not all individuals with RA-specific antibodies progress to clinically manifest RA, the relation between RA-specific antibodies and clonal expansions is unclear. The current data stress once more that the antibodies in the preclinical phase are produced by plasmablasts and long-lived plasma cells located elsewhere (eg, bone marrow and spleen),35 and B cells and plasmablasts present in blood represent migrating cell populations. Moreover, better biomarkers are needed to predict which at-risk individuals will develop RA. Autoantibody and cytokine profiles, specific gene signatures, body mass index, current smoking and autonomic nervous system dysfunction all contribute to the risk of developing RA.36–41 Our data provide a novel biomarker that has superior predictive power compared with other available biomarkers evaluated so far. It increases the accuracy of the previously reported prediction rule for the development of arthritis in autoantibody positive subjects.24

Identification of at-risk individuals who will develop RA in the short term enables development of early preventive strategies.29 42 Our findings support the rationale for B cells or the interaction between B cells and T cells as targets for preventive therapy. The cut-off used here (five or more dominant clones) was chosen to be able to identify subjects with a high risk of developing RA with an acceptable NPV to avoid unnecessary treatment. Whether a preventive pharmacological intervention will be considered acceptable is of course dependent on the benefit/risk profile and the cost-effectiveness of the specific treatment.

As discussed above, there is strong evidence for a role of B cells and plasma cells in the pathogenesis of RA. The development of RA is associated with defects in central and peripheral tolerance leading to autoreactive B cells,43 and circulating autoantibodies can be detected years before the onset of the disease. It is tempting to speculate that the clones identified in the present study are pathogenic B cells since (1) they are not detected in healthy controls nor in subjects at risk who do not develop RA after follow-up, (2) their dominance suggests activity, (3) the clones seem to migrate to the inflamed synovial tissue after arthritis development, and (4) these clones show additional features associated with autoreactivity.

There are technical limitations to the study; first, we measure the BCRheavy chain repertoire on mRNA level since it only identifies expressed BCRs and limits the number of amplifications. However, we cannot distinguish whether clonal signatures are derived from memory B cells or plasma cells. Nevertheless, the presence of dominant BCR clones in the preclinical phase of RA is a robust and reproducible marker. Second, to analyse mRNA, cells were lysed preventing further phenotypic characterisation of dominant clones. Further unravelling the phenotype of BCR clones in the preclinical phase is essential to better understand the role of these cells in the earliest phase of disease, and might lead to even more specific biomarkers. Third, coupling of BCR heavy and light chains is prevented by the technique used, thus limiting determination of reactivity. This should be addressed in a future study.

All patients had clinically manifest arthritis at the time of the second synovial biopsy but the joint that was biopsied was not clinically inflamed except for one patient. Still, these biopsies showed a diverse repertoire resembling the repertoires of clinically inflamed joints, containing the dominant predictive BCR clones identified in peripheral blood in the preclinical phase. This can be explained by the fact that clinically uninvolved joints of patients with established RA exhibit histological signs of synovial inflammation, as described before.44

Taken together, we show the presence of increased BCR clonal signatures in peripheral blood obtained during the preclinical stage of RA. During onset of arthritis, these BCR clones disappear from blood and appear in the target tissue, where they may drive autonomous disease progression. Our observations show that the presence of dominant BCR clones in peripheral blood in the at-risk stage accurately predicts short-term onset of clinically manifest disease. Consequently, they may serve as a biomarker that could help guide decisions about pharmacological treatment to prevent the onset of clinically manifest disease. This is important since recent studies clearly indicate that early intervention may be more effective and lead more often to drug-free remission.45 There are already studies that focus on intervention during the pre-RA phase. An example is the recently completed “PRAIRI” study (http://www.trialregister.nl/trialreg/ admin/rctview.asp?TC=1969), in which individuals at risk of developing RA were treated with a single course of rituximab to delay development of clinically manifest arthritis. The current marker determined during the preclinical phase can be used to further investigate the effect of therapeutical intervention on the clonal distribution over time. Similar studies are currently under way with abatacept and simvastatin. Future work should explore whether BCR clones might also predict onset of disease in other immune-mediated inflammatory disorders like type 1 diabetes, multiple sclerosis, SLE and vasculitis.

Acknowledgments

We would like to thank Rebecca Esveldt, Barbera van Schaik, Marja Jakobs, Ted Bradley and Dr Lisa van Baarsen for technical assistance. This work was carried out on the Dutch national e-infrastructure with the support of SURF Foundation.

Parts of this work have been presented at a scientific meeting and/or published as a conference abstract.

. Epitope spreading of the anti-citrullinated protein antibody response occurs before disease onset and is associated with the disease course of early arthritis. Ann Rheum Dis2010;69:1554–61. doi:10.1136/ard.2009.124537

. The extent of the anti-citrullinated protein antibody repertoire is associated with arthritis development in patients with seropositive arthralgia. Ann Rheum Dis2011;70:128–33. doi:10.1136/ard.2010.132662

. The number of elevated cytokines and chemokines in preclinical seropositive rheumatoid arthritis predicts time to diagnosis in an age-dependent manner. Arthritis Rheum2010;62:3161–72. doi:10.1002/art.27638

. EULAR recommendations for terminology and research in individuals at risk of Rheumatoid arthritis: report from the study group for risk factors for rheumatoid arthritis. Ann Rheum Dis2012;71:638–41. doi:10.1136/annrheumdis-2011-200990

. Expression of the inherently autoreactive idiotope 9G4 on autoantibodies to citrullinated peptides and on rheumatoid factors in patients with early and established rheumatoid arthritis. PLoS One2014;9:e107513. doi:10.1371/journal.pone.0107513

Footnotes

Handling editor Tore K Kvien

Contributors All authors contributed to the formulation of the hypotheses and research questions and the analysis plan, and provided critical input into the draft manuscript. PPT, MJHdH, MHvBT, DVS and DMG were involved in recruitment of patients to the study. MED and PLK performed the experiments under supervision of PPT, FB and NdV. MED, MJHdH, PLK, AHCvK and NdV did the statistical analysis. PPT, MED and NdV drafted the manuscript.

Funding This study was partially supported by the Dutch Arthritis Association (grant no 06-1-303). MED, NdV, DMG and PPT received support from CTMM, the Center for Translational Molecular Medicine, project TRACER (grant 04I-202), and BTCURE, a research project from the Innovative Medicines Initiative Joint Undertaking (grant no 115142-2). PLK was supported by an AMC Graduate School PhD Scholarship.

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